CXCL4L1-fibstatin cooperation inhibits tumor angiogenesis, lymphangiogenesis and metastasis.

Anti-angiogenic and anti-lymphangiogenic drugs slow tumor progression and dissemination. However, an important difficulty is that a tumor reacts and compensates to obtain the blood supply needed for tumor growth and lymphatic vessels to escape to distant loci. Therefore, there is a growing consensus on the requirement of multiple anti-(lymph)angiogenic molecules to stop cell invasion efficiently. Here we studied the cooperation between endogenous anti-angiogenic molecules, endostatin and fibstatin, and a chemokine, the Platelet Factor-4 variant 1, CXCL4L1. Anti-angiogenic factors were co-expressed by IRES-based bicistronic vectors and their cooperation was analyzed either by local delivery following transduction of pancreatic adenocarcinoma cells with lentivectors, or by distant delivery resulting from intramuscular administration in vivo of adeno-associated virus derived vectors followed by tumor subcutaneous injection. In this study, fibstatin and CXCL4L1 cooperate to inhibit endothelial cell proliferation, migration and tubulogenesis in vitro. No synergistic effect was found for fibstatin-endostatin combination. Importantly, we demonstrated for the first time that fibstatin and CXCL4L1 not only inhibit in vivo angiogenesis, but also lymphangiogenesis and tumor spread to the lymph nodes, whereas no beneficial effect was found on tumor growth inhibition using molecule combinations compared to molecules alone. These data reveal the synergy of CXCL4L1 and fibstatin in inhibition of tumor angiogenesis, lymphangiogenesis and metastasis and highlight the potential of IRES-based vectors to develop anti-metastasis combined gene therapies.

Cell-cycle-dependent translational control.

Control of translation in eukaryotes occurs mainly at the initiation step. Translation rates in mammals are robust in the G1 phase of the cell cycle but are low during mitosis. These changes correlate with the activity of several canonical translation initiation factors, which is modulated during the cell cycle to regulate translation.

Translational homeostasis: eukaryotic translation initiation factor 4E control of 4E-binding protein 1 and p70 S6 kinase activities.

Eukaryotic translation initiation factor 4E (eIF4E) is the mRNA 5' cap binding protein, which plays an important role in the control of translation. The activity of eIF4E is regulated by a family of repressor proteins, the 4E-binding proteins (4E-BPs), whose binding to eIF4E is determined by their phosphorylation state. When hyperphosphorylated, 4E-BPs do not bind to eIF4E. Phosphorylation of the 4E-BPs is effected by the phosphatidylinositol (PI) 3-kinase signal transduction pathway and is inhibited by rapamycin through its binding to FRAP/mTOR (FK506 binding protein-rapamycin-associated protein or mammalian target of rapamycin). Phosphorylation of 4E-BPs can also be induced by protein synthesis inhibitors. These observations led to the proposal that FRAP/mTOR functions as a "sensor" of the translational apparatus (E. J. Brown and S. L. Schreiber, Cell 86:517-520, 1996). To test this model, we have employed the tetracycline-inducible system to increase eIF4E expression. Removal of tetracycline induced eIF4E expression up to fivefold over endogenous levels. Strikingly, upon induction of eIF4E, 4E-BP1 became dephosphorylated and the extent of dephosphorylation was proportional to the expression level of eIF4E. Dephosphorylation of p70(S6k) also occurred upon eIF4E induction. In contrast, the phosphorylation of Akt, an upstream effector of both p70(S6k) and 4E-BP phosphorylation, was not affected by eIF4E induction. We conclude that eIF4E engenders a negative feedback loop that targets a component of the PI 3-kinase signalling pathway which lies downstream of PI 3-kinase.

Relief of ornithine decarboxylase messenger RNA translational repression induced by alternative splicing of its 5' untranslated region.

The ornithine decarboxylase enzyme (ODC) is the key regulator of polyamine synthesis and is a member of the cellular proto-oncogene family. Its expression becomes constitutively activated by carcinogens, viruses, and oncogenes. ODC mRNA has a long 5' untranslated region that could be important in the regulation of enzyme levels by affecting translation. To test this hypothesis, we have determined the role of this region on the constitutive ODC hyperexpression measured in AR4-2J cells, an azaserine-induced, tumor-derived pancreatic acinar cell line. Construction of expression vectors in which ODC 5' leader sequence was placed flanking the chloramphenicol acetyltransferase reporter gene allowed us to identify three AR4-2J specific, different alternatively spliced ODC 5' leaders. The 5' ends of exons 2 and 3 were lengthened by 17 and 13 bases, respectively. Translation performed in a cell-free system as well as in COS7 transient transfection experiments demonstrated that AR4-2J isoforms induce a strong increase in the rate of translation. These results provide evidence that alternative splicing observed in tumoral cells, coupled with translation regulation, relieves the translation repression mediated by the long and structured 5' untranslated region of the ODC proto-oncogene.

Essential role for SphK1/S1P signaling to regulate hypoxia-inducible factor 2α expression and activity in cancer.

The sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) signaling pathway has been reported to modulate the expression of the canonical transcription factor hypoxia-inducible HIF-1α in multiple cell lineages. HIF-2α is also frequently overexpressed in solid tumors but its role has been mostly studied in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, where HIF-2α has been established as a driver of a more aggressive disease. In this study, the role of SphK1/S1P signaling with regard to HIF-2α was investigated in various cancer cell models including ccRCC cells. Under hypoxic conditions or in ccRCC lacking a functional von Hippel-Lindau (VHL) gene and expressing high levels of HIF-2α, SphK1 activity controls HIF-2α expression and transcriptional activity through a phospholipase D (PLD)-driven mechanism. SphK1 silencing promotes a VHL-independent HIF-2α loss of expression and activity and reduces cell proliferation in ccRCC. Importantly, downregulation of SphK1 is associated with impaired Akt and mTOR signaling in ccRCC. Taking advantage of a monoclonal antibody neutralizing extracellular S1P, we show that inhibition of S1P extracellular signaling blocks HIF-2α accumulation in ccRCC cell lines, an effect mimicked when the S1P transporter Spns2 or the S1P receptor 1 (S1P1) is silenced. Here, we report the first evidence that the SphK1/S1P signaling pathway regulates the transcription factor hypoxia-inducible HIF-2α in diverse cancer cell lineages notably ccRCC, where HIF-2α has been established as a driver of a more aggressive disease. These findings demonstrate that SphK1/S1P signaling may act as a canonical regulator of HIF-2α expression in ccRCC, giving support to its inhibition as a therapeutic strategy that could contribute to reduce HIF-2 activity in ccRCC.

Gastrin induces phosphorylation of eIF4E binding protein 1 and translation initiation of ornithine decarboxylase mRNA.

Gastrin via its G-protein coupled specific receptor induces transcription of c-fos and c-jun genes through a ras-MAPK pathway. Ornithine Decarboxylase (ODC), a growth regulated proto-oncogene, was chosen to investigate gastrin effects on translation initiation of mRNAs exhibiting a 5'UnTranslated Region (5'UTR) responsible for translation repression in quiescent cells. In AR4-2J tumoral cells, we first demonstrated that gastrin increases ODC mRNA translation. Transient transfections with various CAT chimeric constructs suggested a direct involvement of the 5'UTR in this observation. Translation of this group of mRNAs is enhanced by the availability of the cap-binding protein (eIF4E) that is increased after phosphorylation of its specific binding protein eIF4E-BP1. We found that AR4-2J cells over-expressed eIF4E protein which was not modulated by gastrin treatment. Rapamycin which inhibits 4E-BP1 phosphorylation, completely prevents gastrin-mediated increase of ODC translation indicating that 4E-BP1 could be involved in regulating ODC translation. Implication of 4E-BP1 in mediating gastrin effects is corroborated by the capacity of the ligand to affect 4E-BP1 phosphorylation. These results indicate that gastrin enhances ornithine decarboxylase mRNA translation through a rapamycin sensitive pathway and provide the first evidence in the control of 4E-BP1 phosphorylation after occupancy of a G protein-coupled receptor.

Phosphorylation of the cap-binding protein eIF4E by the MAPK-activated protein kinase Mnk1.

The purpose of this review is to summarize recent experimental data describing the regulation of the phosphorylation of eIF4E, the cap-binding protein, by the MAPK-activated protein kinase Mnk1. Mnk1 does not interact directly with eIF4E, but uses a docking site in eIF4G, a partner of eIF4E. Consequently, control of eIF4E phosphorylation may not strictly depend on changes in Mnk1 activity. The possibility that integrity of the eIF4E/eIF4G/Mnk1 complex also impinges upon eIF4E phosphorylation is discussed.

Pharmacological study of gastrin-mediated amylase release in pancreatic acinar cells (AR4-2J).

In rat pancreatic acinar cells, amylase release and Ca2+ mobilization are related to the occupancy of CCKA receptor. The rat pancreatic acinar cell line (AR4-2J) possesses both CCKA (CCKA R) and CCKB (CCKB R) sub-type receptors. Using this cell line we attempted to determine the relative involvement of each sub-type in both amylase release and Ca2+ mobilization. For this purpose we used L 364718 a selective antagonist for CCKA R and PD 135158 a selective antagonist for CCKB R. We showed on AR4-2J cells that: a minority of CCKA R (Kd = 0.7 nM), a classical CCKB R (Kd = 0.93 nM) and a new high affinity gastrin binding site (Kd = 2.1 pM) coexisted; CCK through CCKA R and CCKB R, was more potent to stimulate amylase secretion (EC50 = 34 pM) and Ca2+ mobilization (EC50 = 30 pM) than to occupy its receptor. Gastrin induced a biphasic stimulation of amylase release. Gastrin through CCKB R was equally potent to stimulate amylase release (EC50 = 1.72 nM) and Ca2+ mobilization (EC50 = 3.1 nM), whereas through the high affinity gastrin binding site, gastrin-induced amylase release (EC50 = 0.73 pM) did not correlate with the Ca2+ mobilization (EC50 = 3.1 nM). These results demonstrated for the first time the existence, on AR4-2J cells, of a high affinity gastrin receptor whose occupation by gastrin induces amylase release.

Pancreatic tumours escape from translational control through 4E-BP1 loss.

The mRNA cap-binding protein eIF4E (eukaryotic translation initiation factor 4E) permits ribosome recruitment to capped mRNAs, and its phosphorylated form has an important role in cell transformation. The oncogenic function of eIF4E is, however, antagonised by the hypophosphorylated forms of the inhibitory eIF4E-binding proteins 1 and 2. eIF4E-binding protein 1 and 2 (4E-BP1 and 2) are two major targets of the protein kinase mTOR, and are essential for the antiproliferative effects of mTOR inhibitors. Herein, we report that pancreas expresses specifically and massively 4E-BP1 (4E-BP2 is nearly undetectable). However, 4E-BP1 expression is extinguished in more than half of the human pancreatic ductal adenocarcinomas (PDAC). 4E-BP1 shutoff is recapitulated in a mouse genetic model of PDAC, which is based on a pancreas-specific mutation of Kras, the more frequently mutated oncogene in human pancreatic tumours. 4E-BP1 downregulation enhances eIF4E phosphorylation and facilitates pancreatic cancer cell proliferation in vitro and tumour development in vivo. Furthermore, 4E-BP1 loss combined with the absence of 4E-BP2 renders eIF4E phosphorylation, protein synthesis and cell proliferation resistant to mTOR inhibition. However, proliferation can be better limited by a recently developed compound that mimics the function of 4E-BP1 and 2 independently of mTOR inhibition.

Dual roles of hemidesmosomal proteins in the pancreatic epithelium: the phosphoinositide 3-kinase decides.

Given the failure of chemo- and biotherapies to fight advanced pancreatic cancer, one major challenge is to identify critical events that initiate invasion. One priming step in epithelia carcinogenesis is the disruption of epithelial cell anchorage to the basement membrane which can be provided by hemidesmosomes (HDs). However, the existence of HDs in pancreatic ductal epithelium and their role in carcinogenesis remain unexplored. HDs have been explored in normal and cancer pancreatic cells, and patient samples. Unique cancer cell models where HD assembly can be pharmacologically manipulated by somatostatin/sst2 signaling have been then used to investigate the role and molecular mechanisms of dynamic HD during pancreatic carcinogenesis. We surprisingly report the presence of mature type-1 HDs comprising the integrin α6ß4 and bullous pemphigoid antigen BP180 in the human pancreatic ductal epithelium. Importantly, HDs are shown to disassemble during pancreatic carcinogenesis. HD breakdown requires phosphoinositide 3-kinase (PI3K)-dependent induction of the matrix-metalloprotease MMP-9, which cleaves BP180. Consequently, integrin α6ß4 delocalizes to the cell-leading edges where it paradoxically promotes cell migration and invasion through S100A4 activation. As S100A4 in turn stimulates MMP-9 expression, a vicious cycle maintains BP180 cleavage. Inactivation of this PI3K-MMP-9-S100A4 signaling loop conversely blocks BP180 cleavage, induces HD reassembly and inhibits cell invasion. We conclude that mature type-1 HDs are critical anchoring structures for the pancreatic ductal epithelium whose disruption, upon PI3K activation during carcinogenesis, provokes pancreatic cancer cell migration and invasion.

Anti-oncogenic potential of the eIF4E-binding proteins.

The eIF4E-binding proteins (4E-BPs) are inhibitors of protein synthesis that sequester the mRNA cap-binding protein eIF4E and consequently block cell growth and proliferation. In most tumors however, their inhibitory function is compromised by major oncogenic signaling pathways. Recently, thanks to the generation of mouse genetic models, considerable progress has been made in elucidating the involvement of 4E-BPs and their unique target, eIF4E, in the process of carcinogenesis. Increasing evidence indicates that an 'addiction' to protein synthesis emerges in cancer cells, highlighting the potential that 4E-BPs have as targets for therapeutics. In this review, we summarize the biochemical function, regulation and anti-oncogenic activity of the 4E-BPs.

Phosphatidylinositol 3-kinase-dependent transcriptional silencing of the translational repressor 4E-BP1.

The suppressor of translation initiation 4E-BP1 functions as a key regulator in cellular growth, differentiation, apoptosis and survival. While the control of 4E-BP1 activity via phosphorylation has been widely studied, the molecular mechanisms and the signaling pathways that govern 4E-BP1 gene expression are largely unknown. Here we show that inactivation of phosphatidylinositol 3-kinase (PI3K) consequent to stable expression of the antiproliferative somatostatin receptor 2 (sst2) in pancreatic cancer cells leads to transcriptional accumulation of the hypophosphorylated forms of 4E-BP1 protein. In cancer cells, while 4E-BP1 gene promoter is maintained repressed in a PI3K-dependent mechanism, sst2-dependent inactivation of the PI3K/Akt pathway releases 4E-BP1 gene transcription. Furthermore, the use of a pharmacological inhibitor and dominant-negative or -positive mutants of PI3K all affect 4E-BP1 protein expression and promoter activity in different cell lines. These data show that, in addition to inactivation of 4E-BP1 via hyperphosphorylation, signaling through the PI3K pathway silences 4E-BP1 gene transcription.

Alternative splicing facilitates internal ribosome entry on the ornithine decarboxylase mRNA.

Ornithine decarboxylase (ODC) is the ratelimiting enzyme in the biosynthesis of polyamines, which are required for optimal cell growth and proliferation. ODC is overexpressed in many tumors and, conversely, its overexpression induces transformation. We have previously reported that ODC mRNA alternative splicing relieves the translation repression normally imposed by a long and structured 5' untranslated region (UTR), and that the ODC 5' UTR contains an internal ribosome entry site (IRES). Here we show that ODC IRES activity is enhanced following inclusion of alternative sequences generated by splicing at cryptic acceptor sites. Furthermore, the alternative ODC IRES is more sensitive to cell-cycledependent changes in the rate of translation. These findings uncover a new biological property of differentially spliced transcripts. This is the first example of alternative splicing that modulates mRNA translation through the cell cycle in a cap-independent manner.

Physiology of somatostatin receptors.

Since its discovery three decades ago as an inhibitor of GH release from the pituitary gland, somatostatin has attracted much attention because of its functional role in the regulation of a wide variety of physiological functions in the brain, pituitary, pancreas, gastrointestinal tract, adrenals, thyroid, kidney and immune system. Its actions include inhibition of endocrine and exocrine secretions, modulation of neurotransmission, motor and cognitive functions, inhibition of intestinal motility, absorption of nutrients and ions and vascular contractility. In addition, the peptide controls the proliferation of normal and tumor cells. Its action is mediated by a family of G protein-coupled receptors [somatostatin receptor (SSTR)1-SSTR5] that are widely distributed in normal and cancer cells. Direct antitumor activities, mediated through SSTR expressed in tumor cells, include blockade of autocrine/paracrine growth-promoting hormone and growth factor production, inhibition of growth factor-mediated mitogenic signals and induction of apoptosis. Indirect antitumor effects include inhibition of growth-promoting hormone and growth factor secretion, and antiangiogenic actions. Many human tumors express more than one SSTR subtype, with SSTR2 being predominant. These receptors represent the molecular basis for the clinical use of somatostatin analogs in the treatment of endocrine tumors and their in vivo localization. This review covers the present knowledge in SSTR biology and signaling.

Suppression of cap-dependent translation in mitosis.

Cap-dependent translation is mediated by eIF4F, a protein complex composed of three subunits as follows: eIF4E, which recognizes the mRNA 5' cap structure; eIF4A, an RNA-helicase; and eIF4G, a scaffolding protein that binds eIF4E, eIF4A, and the eIF4E-kinase Mnk1 simultaneously. eIF4E is hypophosphorylated and cap-dependent translation is reduced at mitosis. Here, we show that 4E-BP1, a suppressor of eIF4E function, is also hypophosphorylated in mitosis, resulting in disruption of the eIF4F complex. Consequently, eIF4E is sequestered from the eIF4G/Mnk1 complex. These results explain the specific inhibition of cap-dependent translation in mitosis and also explain how eIF4E is rendered hypophosphorylated during mitosis. Furthermore, eIF4E interaction with eIF4GII is strongly decreased coincident with hyperphosphorylation of eIF4GII. Thus, inhibition of cap-dependent translation in mitosis results from a combination of phosphorylation modifications leading to eIF4F complex disruption.

Irresistible IRES. Attracting the translation machinery to internal ribosome entry sites.

Studies on the control of eukaryotic translation initiation by a cap-independent recruitment of the 40S ribosomal subunit to internal messenger RNA sequences called internal ribosome entry sites (IRESs) have shown that these sequence elements are present in a growing list of viral and cellular RNAs. Here we discuss their prevalence, mechanisms whereby they may function and their uses in regulating gene expression.

A cell cycle-dependent internal ribosome entry site.

The eukaryotic mRNA 5' cap structure facilitates translation. However, cap-dependent translation is impaired at mitosis, suggesting a cap-independent mechanism for mRNAs translated during mitosis. Translation of ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, peaks twice during the cell cycle, at the G1/S transition and at G2/M. Here, we describe a cap-independent internal ribosome entry site (IRES) in the ODC mRNA that functions exclusively at G2/M. This ensures elevated levels of polyamines, which are implicated in mitotic spindle formation and chromatin condensation. c-myc mRNA also contains an IRES that functions during mitosis. Thus, IRES-dependent translation is likely to be a general mechanism to synthesize short-lived proteins even at mitosis, when cap-dependent translation is interdicted.

Regulation of SOCS-1 expression by translational repression.

Accumulating evidence demonstrates that cytokine receptor signaling is negatively regulated by a family of Src homology 2 domain-containing adaptor molecules termed SOCS (suppressor of cytokine signaling). Previous studies have indicated that the expression of SOCS-related molecules is tightly controlled at the level of transcription. Furthermore, it has been reported that SOCS polypeptides are relatively unstable in cells, unless they are associated with elongins B and C. Herein, we document the existence of a third mechanism of regulation of SOCS function. Our data showed that expression of SOCS-1, a member of the SOCS family, is strongly repressed at the level of translation initiation. Structure-function analyses indicated that this effect is mediated by the 5' untranslated region of socs-1 and that it relates to the presence of two upstream AUGs in this region. Further studies revealed that socs-1 translation is cap-dependent and that it is modulated by eIF4E-binding proteins. In combination, these results uncover a novel level of regulation of SOCS-related molecules. Moreover, coupled with previous findings, they suggest that SOCS expression is tightly regulated through multiple mechanisms, in order to avoid inappropriate interference with cytokine-mediated effects.